Wolbachia prevalence in the vector species Culex pipiens and Culex torrentium in a Sindbis virus-endemic region of Sweden

Background Wolbachia pipientis are endosymbiotic bacteria present in a large proportion of terrestrial arthropods. The species is known to sometimes affect the ability of its host to transmit vector-borne pathogens. Central Sweden is endemic for Sindbis virus (SINV), where it is mainly transmitted by the vector species Culex pipiens and Culex torrentium, with the latter established as the main vector. In this study we investigated the Wolbachia prevalence in these two vector species in a region highly endemic for SINV. Methods Culex mosquitoes were collected using CDC light traps baited with carbon dioxide over 9 years at 50 collection sites across the River Dalälven floodplains in central Sweden. Mosquito genus was determined morphologically, while a molecular method was used for reliable species determination. The presence of Wolbachia was determined through PCR using general primers targeting the wsp gene and sequencing of selected samples. Results In total, 676 Cx. pipiens and 293 Cx. torrentium were tested for Wolbachia. The prevalence of Wolbachia in Cx. pipiens was 97% (95% CI 94.8–97.6%), while only 0.7% (95% CI 0.19–2.45%) in Cx. torrentium. The two Cx. torrentium mosquitoes that were infected with Wolbachia carried different types of the bacteria. Conclusions The main vector of SINV in the investigated endemic region, Cx. torrentium, was seldom infected with Wolbachia, while it was highly prevalent in the secondary vector, Cx. pipiens. The presence of Wolbachia could potentially have an impact on the vector competence of these two species. Furthermore, the detection of Wolbachia in Cx. torrentium could indicate horizontal transmission of the endosymbiont between arthropods of different species. Graphical abstract


Background
The transmission of arboviruses is influenced by a number of factors, including both abiotic (e.g., temperature) and biotic elements (e.g., vector immune status) [1][2][3][4]. One important biotic factor is the intracellular symbiont Wolbachia pipientis (Class: Alphaproteobacteria, Order: Rickettsiales), present in some nematode species and an estimated 40% of all terrestrial arthropods [5]. Wolbachia is a genetically diverse species, composed of 18 phylogenetically distinct supergroups described to date (A-R) [6]. Deeply involved in the reproduction of its host [7], Wolbachia is known for inducing cytoplasmic incompatibility and giving rise to crossing types, most studied in Culex pipiens and its Wolbachia strain wPip, which belongs to supergroup B [8][9][10]. Additionally, it is well established that Wolbachia infection in mosquitoes can influence their ability to become infected and transmit several arboviruses [11][12][13][14].
Culex torrentium is regarded as the most important enzootic vector in Sweden due to its high abundance in endemic areas, high infection rate, and superior vector competence to Cx. pipiens [31][32][33][34]. One difference between Cx. torrentium and Cx. pipiens is the prevalence of Wolbachia-infected individuals. Previous studies in Germany, Belgium, Russia, Belarus, Kazakhstan, and Kyrgyz Republic have found Wolbachia to be very common in Cx. pipiens but absent in Cx. torrentium [35][36][37][38]. It is therefore possible that these differences in Wolbachia infection status could account for part of the difference in vector competence seen between Cx. pipiens and Cx. torrentium. Previous studies have however only been performed in regions without intense SINV transmission. Therefore, this study aims at investigating the Wolbachia prevalence in Cx. pipiens and Cx. torrentium collected in a highly SINV-endemic region in central Sweden.

Mosquitoes
Mosquitoes were collected at 50 different locations across the River Dalälven floodplains (Fig. 1) as part of a routine mosquito monitoring programme [39]. SINV is considered endemic to this region and some of the highest infection rates in mosquitoes have been detected here [22,33]. Collections were performed every second week between May and September during the years 2010-2018 using CDC light traps baited with carbon dioxide. Mosquitoes were identified based on morphological characteristics [40], and Cx. pipiens/torrentium were sorted out and used for molecular identification to species. Briefly, individual mosquitoes were homogenized in 500 µl of phosphate-buffered saline (PBS) supplemented with 20% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B (Thermo Fischer Scientific; Waltham, MA, USA) using two steel beads in the Qiagen TissueLyser II ™ (Qiagen; Hilden, Germany). Five microliters (5 µl) of the homogenate was pretreated by incubating at 98 °C for 2 min in 20 µl of dilution buffer with 0.5 µl of DNA release additive, part of the Phire Tissue Direct PCR Master Mix kit (Thermo Scientific; Vilnius, Lithuania). The pretreated homogenate was stored at −20 °C before being used as a template in polymerase chain reaction (PCR).
Conventional PCR of part of the cytochrome oxidase subunit I (COI) was performed in 20 µl reactions with 1 µl template using the forward primer C1-J-2183 (5′-CAA CAT TTA TTT TGA TTT TTTGG-3′) and the reverse primer TL2-N-3014 (5′-TCC AAT GCA CTA ATC TGC CAT ATT A-3′) at a concentration of 0.5 µM each under the following thermocycler conditions: initial denaturation at 98 °C for 5 min, followed by 40 cycles of denaturation at 98 °C for 5 s, annealing at 54.5 °C for 5 s and extension at 72 °C for 20 s, and a final extension step at 72 °C for 1 min. A PCR-restriction fragment length polymorphism (PCR-RFLP) assay [41] was performed on the PCR product, using the restriction enzymes FspBi and SspI (Thermo Fischer Scientific; Vilnius, Lithuania).
Culex pipiens molestus mosquitoes, originating from a field population sampled in Gothenburg, Sweden [42] and reared in our in-house mosquito rearing facility, were used as positive controls in PCR as they are naturally infected with a wPip strain of Wolbachia (data not shown). These were also used for PCR optimization.

Wolbachia detection
Wolbachia detection was performed through PCR on 5 µl of the mosquito homogenate, using the same Tissue Direct kit procedures as described above. Wolbachia primers 81F (5′-TGG TCC AAT AAG TGA TGA AGA AAC -3′) and 691R (5′-AAA AAT TAA ACG CTA CTC CA-3′), designed for general detection of Wolbachia within supergroups A and B [43], were used at a final concentration of 0.5 µM each. The thermocycler conditions for Wolbachia detection were as described above but with the annealing temperature set to 58 °C. A subset of samples was also tested with a confirmatory PCR to determine whether the detected wsp gene Wolbachia belonged to that of the wPip strain using wPipspecific primers wPF (5′-CGA CGT TAG TGG TGC AAC ATTTA-3′) and wPR (5′-AAT AAC GAG CAC CAG CAA AGAGT-3′) [44] with the same PCR conditions as described previously but with the annealing temperature set to 56 °C. For primer optimization, DNA integrity was controlled by extraction of total DNA to make sure that a negative PCR result was not due to DNA degradation in the sample. DNA was extracted from 44 samples with the E.Z.N.A. ® Tissue DNA Kit (Omega Bio-Tek, Inc., Norcross, GA, USA), and visual inspection of DNA integrity was done by gel electrophoresis. Extracted DNA and all PCR products were visualized on 1.8% agarose gel stained with GelRed ® Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA) (Fig. 2). A subset of PCR products was purified with ExoSAP-IT ® (Thermo Fischer Scientific; Vilnius, Lithuania) and sequenced through Sanger sequencing (Macrogen; Amsterdam, The Netherlands) to validate the method and verify the results.

Data analysis
All records were kept and analysed in Microsoft Excel 2016 (Microsoft; Redmond, CA, USA). Confidence intervals for Wolbachia prevalence were calculated assuming binomial distribution using the Wilson score interval through RStudio (RStudio team, Boston, USA). P-values to determine statistical significance for differences in Wolbachia prevalence between years were calculated using Fisher's exact test with Bonferroni correction. Sequences of PCR fragments were analysed in the BioEdit sequence alignment editor version 7.2.5 [45].

Discussion
We found that Wolbachia was highly prevalent in Cx. pipiens collected around the River Dalälven floodplains, while it was nearly absent from Cx. torrentium. This is in line with previous European studies investigating large samples of Cx. pipiens, with reported Wolbachia prevalence of 91% in western Russia [35], 95% in central Russia, 81% in Belarus [36], and 93% in Germany [37]. Raharimalala et al. [38] detected Wolbachia in nine out of nine tested adult Cx. pipiens and 26 out of 48 larvae collected in Belgium, which also supports the generally high prevalence of Wolbachia in this species. Interestingly, the different populations studied by Khrabrova et al. [36] had varying levels of Wolbachia prevalence, some with as few as 34.5% of individuals carrying the endosymbiont. Wolbachia is reported to approach fixation in most Cx. pipiens populations worldwide [46,47], but this does not seem to hold true for all European populations.
Only Ricci et al. [48] have, to our knowledge, previously found Wolbachia in Cx. torrentium, after testing only two individuals collected in Italy. Raharimalala et al. [38], Leggewie et al. [37], Vinogradova et al. [35], and Khrabrova et al. [36] detected no Wolbachia in Cx. torrentium despite having tested 42 Belgian, 188 German, 321 Russian, and 853 Eastern European individuals, respectively. Our study, as well as the study by Ricci et al. [47], tested adult mosquitoes, while the four that failed to detect Wolbachia in Cx. torrentium tested field-collected larvae and pupae. Wolbachia is usually inherited and should thus be present in all life stages of the mosquito; however, life stage is still potentially an important consideration when screening for Wolbachia, both to avoid analysing siblings and to detect potential horizontal transmission.
Due to the low prevalence of Wolbachia in Cx. torrentium, we hypothesize that the two positive individuals or their recent ancestors acquired the infection horizontally. Transmission could potentially have occurred by feeding on the same plants as other arthropods [49,50] or through arthropod parasites, such as through mites sometimes feeding on mosquitoes [51,52]. Despite wsp being a poor marker of Wolbachia strain due to its tendency to recombine [53], the lineage of the Cx. torrentium whose wsp gene matched that of the wPip strain could have acquired its infection from a Cx. pipiens through their shared habitat and ecological niche. Alternative sources are also possible, since a highly similar wsp sequence has also been found in other Palearctic insects. Further studies on the mechanisms for horizontal Wolbachia transmission involving mosquitoes are needed to fully explain the occasional spread of Wolbachia to Cx. torrentium.
The restriction of SINV outbreaks to Northern Europe has been suggested to be connected to the relatively higher abundance of the competent vector species Cx. torrentium in SINV-endemic regions [31,32]. Under laboratory conditions, Cx. torrentium is significantly more susceptible to SINV infection than Cx. pipiens [34,54]. The presence of Wolbachia in Cx. pipiens may contribute to its lower susceptibility to SINV. Such reduction in vector competence is often seen when transferring a novel Wolbachia strain into a mosquito species that is naturally Wolbachia-free or naturally carries a different strain [11,[55][56][57][58], but the impact of a naturally occurring Wolbachia infection (i.e., native infection) is not as clear, with reports of both reduced vector competence [13,14,59] and no observed effect [60][61][62][63]. No vector competence studies have been done on the role of Wolbachia in alphavirus transmission in Culex mosquitoes. With relatively few data to extrapolate from, empirical investigation is needed to evaluate the impact of Wolbachia on the SINV transmission cycle.

Conclusions
Our study, performed in a SINV-endemic region of Sweden, confirmed previously reported general patterns of Wolbachia infection in Culex mosquitoes, with most Cx. pipiens and very few Cx. torrentium carrying the endosymbiont, which potentially has implications for their differences in vector competence. Our findings, paired with the specific conditions under which SINV is transmitted in Sweden, prompt more research into Wolbachia's role in the SINV transmission cycle as well as the horizontal routes of Wolbachia transmission among mosquitoes.

Funding
Open access funding provided by Uppsala University. The research was funded by the Swedish Society for Medical Research, the Carl Trygger Foundation, and the European Union's Horizon 2020 research and innovation programme under Grant No. 874735 (VEO). None of the funding bodies had any role in the design of the study or collection, analysis, and interpretation of data, or in drafting of the manuscript.

Availability of data and materials
For amplicon sequences of the Wolbachia detected in Cx. torrentium and Cx. pipiens, the data sets generated and/or analysed during the current study are available in the GenBank repository, accession numbers MW622245-MW622247. All other data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.